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What if DNA extension could be terminated at a known nucleotide using a mixture of normal bases and termination bases TACGTACGTACGTGT ATGCATGCATGC???????????????????????????????????? 3’ 5’ Primer DNA Pol. A TACGTACGTACGTGT CG ATGCATGCATGC???????????????????????????????????? 3’ 5’ Primer A DNA Pol. A Normal base gets incorporated By probability termination will occur at every “A”

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PhD 1943 Cambridge University Nobel Prize In Chemistry 1958 Amino acid sequence of insulin Nobel Prize In Chemistry 1980 Sequenced the first genome, phage Φ-X174, by hand using a method that he developed. Frederick Sanger The Sanger Dideoxy sequencing method was the foundation for the discovery of PCR.

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“…no one was particularly enthusiastic about it.” 1984 annual Cetus Scientific Meeting…..”nobody seemed to be interested in my poster….” “People would glance at it and keep walking.” At first, people did not get it. Then Joshua Lederberg (also a Nobel Laureate) said: “Why didn’t I think of that?”

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1993 Nobel Prize

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So, how is PCR important to your life, right now? Many SNP’s are associated with disease. Do you have a risk allele?

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First, you need to select primers for PCR No, you do not need a computer program to select primers. I prefer 24 bp long and end on G or C Others prefer 20 bp long and end on A or T Try to have at least 50% G’s +C’s to ensure reasonable annealing temperature That’s about it. Do not waste too much time selecting primers.

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New Thermocyclers = Peltier Cooling & Gradient Blocks Peltier effect It occurs when a current is passed through two dissimilar metals or semiconductors (n-type and p- type) that are connected to each other at two junctions (Peltier junctions). The current drives a transfer of heat from one junction to the other: one junction cools off while the other heats up; as a result, the effect is often used for thermoelectric cooling. This effect was observed in 1834 by Jean Peltier.

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RT-PCR measures the presence of cDNA corresponding to its respective RNA. RT-PCR is, therefore, used to indirectly estimate RNA abundance which MAY indicate the level of gene expression.

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Major Points Sequencing and PCR both use DNA polymerase and replicate DNA (PCR uses TAQ DNA polymerase). Kary Mullis discovered PCR while thinking about a possible dideoxy sequencing experiment. Half of a Nobel discovery is finding it, the other half is realizing what you have found. RT-PCR is used to estimate gene expression